Staining For Thrombospondin-1 Research Articles

Expression of thrombospondin-1 in conjunctival squamous cell carcinoma is correlated to the Ki67 index and associated with progression-free survival.

Conjunctival squamous cell carcinoma (SCC) is primarily treated with surgical resection. SCC has various stages, and local recurrence is common. The purpose of this study was to investigate thrombospondin-1 expression and its association with prognosis. In this retrospective study, a gene expression array along with immunohistochemistry were performed for the evaluation of thrombospondin-1 expression, localization, as well as Ki67 labeling cell indices in carcinoma in situ (Tis) and advanced conjunctival SCC (Tadv). The presence or absence and intensity of cytoplasmic and nuclear staining in tumor cells were also divided into groups with a score of 0-3 and semi-quantitatively analyzed to investigate intracellular staining patterns. The association between thrombospondin-1 expression and tumor progression in a series of 31 conjunctival SCCs was further investigated. All 31 patients in the cohort (100%) were East Asian. A simple comparison between Tis and Tadv demonstrated significant differences in expressions of 45 genes, including thrombospondin-1 (p < 0.01). In this cohort, 30/31 tumors were positive (96%) for thrombospondin-1. Furthermore, thrombospondin-1 intracellular staining pattern analysis scores were 2.12 and 0.96 for nuclear and cytoplasmic staining, respectively, with a significant difference observed between Tis and Tadv (p < 0.01). Alteration of the Ki67 labeling index was significantly correlated with that of the thrombospondin-1 cytoplasmic score (p = 0.030). Furthermore, univariate Cox regression analysis showed a significant correlation between thrombospondin-1 staining and progression-free survival (p = 0.026) and final orbital exenteration (p = 0.019). The present results demonstrated that thrombospondin-1 is a potential molecular target in the pathology of conjunctival SCC, in addition to serving as a prognostic factor.

Investigation of thrombospondin 1 (THBS1) as prognostic and predictive factor of neoadjuvant chemotherapy for advanced gastric cancer.

49 Background: Curative R0 operation is the best treatment for patients with Stage II/III gastric cancer, but still there are recurrent cases. Although chemotherapy is an important treatment, there is no evidence of neoadjuvant chemotherapy (NAC) for both R0 operated advanced gastric cancer and inoperable cases. Thrombospondin 1 (THBS1) plays a role in angiogenesis in many cancers, and we reported that THBS1 could be prognostic factor of Stage IV gastric cancer and predictive factor of S-1+ paclitaxel chemotherapy. The aim of this study is to investigate THBS1 plays as prognostic and predictive factor of neoadjuvant chemotherapy for advanced gastric cancer. Methods: Study 1. Investigation of THBS1 as prognostic factor in R0 cases: 129 patients with R0 operated for stage II/III gastric cancer (II: 56, III: 73) were enrolled in this study. THBS1 expression was investigated by immunostaining with resected specimen. Relationship between THBS1 expression and disease free survival (DFS), and recurrence rate were evaluated. Study 2. Investigation of THBS1 as predictive factor in unresectable cases: Among 111 patients who had unresectable gastric cancer, 55 patients had NAC (NAC (+) group; S-1+PTX i.p.: 23, Docetaxel+CDDP+S-1 (DCS): 17, S-1+CDDP 6, Docetaxel{S-1 (DS): 5, S-1 alone: 4) were compared with NAC (-) group (n=56) by evaluation of relationship between immunohistological expression of THBS1 and overall survival. Results: Study 1: There were 39 (30%) gastric cancers with positive staining for THBS1, 19 patients (49%) recurred cancer and 5-year DFS was 20% in THBS (+) group. Among 90 patients of THBS (-) group, 19 patients (20%) recurred, and DFS was 73%. Study 2: there were 32 (29%) unresectable gastric cancers with THBS1 (+). NAC (+) group showed 30.4% of 2-year OS, whereas 13.1% of NAC (-) group. Among NAC (+) group, 24 (43.6%) patients showed response of NAC, and among responder group, 13 (54%) patients showed THBS1 (+), whereas 10 (28.6%) patients showed THBS (+) in non-responder group. Conclusions: THBS1 expression showed reverse correlation with prognosis and positive correlation with prediction of neoadjuvant chemotherapy for Stage II/III gastric cancer.

Expression of thrombospondin-1 and Ski are prognostic factors in advanced gastric cancer

It has been suggested that thrombospondin-1 (TSP-1) plays a role in angiogenesis in many cancers. In addition, TSP-1 has been shown to suppress tumor growth by activating transforming growth factor-β (TGF-β). Recent studies have shown that Ski protein suppresses TGF-β signaling. The aim of this study was to investigate the role of TSP-1 and Ski expression in advanced gastric cancer. Sixty-five recurrent patients who underwent resection of advanced gastric cancer (n=62) or who had unresectable tumors (n=3) between March 2003 and October 2007 participated in this study. All patients were treated with chemotherapy after collection of the tumor tissue. Twenty-three of them were also treated with chemotherapy before collection of the tumor specimen. The TSP-1, Ski, vascular endothelial growth factor (VEGF) and TGF-β protein expression were examined by immunohistochemical staining, and microvessels in the tumor were counted. There were 17 (26.2%) gastric cancers with positive staining for TSP-1. There was no relationship between TSP-1 expression and clinicopathological factors. TSP-1 expression had positive association with VEGF expression, microvessel density (MVD) and TGF-β expression. However, there was no relationship between TSP-1 and Ski expression. TSP-1 correlated with good survival in advanced gastric cancer, and Ski correlated with poor survival in the patients with TGF-β-positive advanced gastric cancer. This study shows that TSP-1 enhances angiogenesis due to its positive correlation with VEGF and MVD, and is a good prognostic factor for survival in advanced gastric cancer. This study also shows that Ski inhibits the prolonged effects of TGF-β for survival in advanced gastric cancer.

Correlation between the expression of thrombospondin-1 and neovascularization in mucoepidermoid carcinoma

Researchers have recently demonstrated that thrombospondin-1 (TSP-1) has an important function in regulating neovascularization. Whether it inhibits or accelerates neovascularization, however, is still controversial. We found few reports about the correlation between TSP-1 and vascularization in mucoepidermoid carcinoma. In this research, the distribution and expression of TSP-1 in mucoepidermoid carcinoma were investigated. We also analyzed (1) the correlation between the expression of TSP-1 and microvessel density (MVD), as an indicator of neovascularization activity, and (2) the effect of TSP-1 on neovascularization and tumor growth in the subcutaneous xenotransplanted model of mucoepidermoid carcinoma. (1) The sites and intensity of expression of TSP-1 and the MVD were analyzed in 45 cases of mucoepidermoid carcinoma after surgery by the method of streptavidin-peroxidase (SP) immunohistochemistry; and (2) recombinant human thrombospondin-1 (rhTSP-1) was injected twice a week for five consecutive weeks around the tumor in the subcutaneous xenotransplanted tumor model of mucoepidermoid carcinoma in nude mice. Each week, the tumor size was measured, in order to draw the growth curve of the xenotransplanted tumor model of mucoepidermoid carcinoma, and MVD was measured. (1) The positive expression of TSP-1 protein was 57.78% (26/45). Most positive staining for TSP-1 was found in the cytoplasm of the cancer cells, while some staining occurred in the extracellular matrix. The mean MVD in 45 cases of mucoepidermoid carcinoma was 58.17 +/- 19.77 per 100 visual fields. Tumors with a high expression of TSP-1 showed a low MVD value, and the TSP-1 immunocompetence and microvessel density showed a significant negative correlation (r(s) = -0.947, P < 0.001). (2) The xenotransplanted tumors with the injection doses of 1.25, 0.75 and 0.25 microg/ml respectively were 36.97%, 53.36% and 73.61% of the size of the control group ((451 +/- 92), (651 +/- 113), (898 +/- 86) and (1220 +/- 157) mm(3) respectively, F = 53.167, P < 0.001), and their weights were respectively 35.14%, 51.35% and 70.27% of the control group ((1.3 +/- 0.5), (1.9 +/- 0.5), (2.6 +/- 0.3), and (3.7 +/- 0.7) g respectively, F = 62.669, P < 0.001). Their MVDs were 25.00%, 45.93%, and 72.20% respectively of the control group and concentration dependent (15.43 +/- 3.45, 28.35 +/- 4.24, 44.57 +/- 3.35 and 61.73 +/- 5.43 per 100 visual fields respectively, F = 54.582, P < 0.001). The TSP-1 has a higher expression in mucoepidermoid carcinoma and the expression has a significant negative correlation with neovascularization. The TSP-1 inhibits neovascularization and tumor growth, and it might be a new biological therapy for treatment of patients with mucoepidermoid carcinoma.

Angiogenesis in pterygium: study of microvessel density, vascular endothelial growth factor, and thrombospondin-1

This retrospective study aims to elucidate the role of angiogenesis in the pathogenesis of pterygium. We evaluated microvessel density (MVD), and expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Fifty-two surgically excised pterygia and seven normal conjunctivae were immunohistochemically studied applying the streptavidin-biotin method in paraffin-embedded tissue sections. Monoclonal antibodies were targeted against CD31, VEGF, and TSP-1 proteins. Pterygium presented with statistically significant higher average count of microvessels compared to normal conjunctivae (17.97+/-8.5 vs5.72+/-5 per high power field, P=0.001). In 24/52 (46.2%) cases of pterygium, high expression levels for VEGF were demonstrated, whereas the mean percentage of VEGF-positive epithelial cells was 58.03%. Furthermore, normal conjunctival presented statistically significant higher expression levels for VEGF in epithelial cells (83.14+/-36.08 vs58.03+/-31.23%, P=0.007). On the contrary, the presence of VEGF immunoreactivity in vascular endothelial and stromal cells was significantly higher in pterygium tissues (P<0.0001). Stromal staining for TSP-1 was detected in only 29/52 (55.8%) of the cases and no correlation with normal conjunctivae was found. Finally, statistically significant positive correlation between MVD values and stromal VEGF expression was found (P=0.049). The angiogenesis-related factors that were studied proved to be highly expressed in pterygium tissue. On the contrary, TSP expression level was low, allowing inducers of angiogenesis to act uninhibited. This phenomenon could provide the pathogenic basis of pterygium formation.

Clinicopathologic study of vascular endothelial growth factor, thrombospondin-1, and microvessel density assessed by CD34 in patients with stage III ovarian carcinoma

The aim of the study was to investigate angiogenesis in patients with advanced-stage ovarian carcinoma. We used paraffin-embedded tumor tissues from 33 patients diagnosed with FIGO III ovarian cancer who had optimal surgery and received platinum-based chemotherapy. The tissue expression of CD34, vascular endothelial growth factor (VEGF), and thrombospondin-1 (TSP-1) was assessed immunohistochemically. CD34 stained hot spot areas were used to evaluate tumor microvessel density (MVD). VEGF and TSP-1 were assessed by semiquantitative methods. The studied molecules were investigated for relationship with standard clinicopathologic parameters. MVD count was high: median value of 39, range 12-143 microvessels/mm2. VEGF was present in all cases and stained strong in 91%. Stroma staining for TSP-1 was weak in 79% of the cases, strong in 6%, and absent in five (15%). We did not find correlations between the three studied markers and histologic type or tumor grade. MVD score did not relate to VEGF or TSP-1. We only observed a trend toward a longer survival in patients with tumors expressing high TSP-1 (60 vs. 36 months, P= 0.1). Proangiogenetic factor VEGF is highly expressed in advanced-stage ovarian carcinomas. The findings of this study may offer support for considering VEGF-targeted therapeutics in ovarian cancer treatment research.

Clinicopathologic study of vascular endothelial growth factor, thrombospondin-1, and microvessel density assessed by CD34 in patients with stage III ovarian carcinoma

The aim of the study was to investigate angiogenesis in patients with advanced-stage ovarian carcinoma. We used paraffin-embedded tumor tissues from 33 patients diagnosed with FIGO III ovarian cancer who had optimal surgery and received platinum-based chemotherapy. The tissue expression of CD34, vascular endothelial growth factor (VEGF), and thrombospondin-1 (TSP-1) was assessed immunohistochemically. CD34 stained hot spot areas were used to evaluate tumor microvessel density (MVD). VEGF and TSP-1 were assessed by semiquantitative methods. The studied molecules were investigated for relationship with standard clinicopathologic parameters. MVD count was high: median value of 39, range 12–143 microvessels/mm2. VEGF was present in all cases and stained strong in 91%. Stroma staining for TSP-1 was weak in 79% of the cases, strong in 6%, and absent in five (15%). We did not find correlations between the three studied markers and histologic type or tumor grade. MVD score did not relate to VEGF or TSP-1. We only observed a trend toward a longer survival in patients with tumors expressing high TSP-1 (60 vs. 36 months, P= 0.1). Proangiogenetic factor VEGF is highly expressed in advanced-stage ovarian carcinomas. The findings of this study may offer support for considering VEGF-targeted therapeutics in ovarian cancer treatment research.

Impairment of thrombospondin-1 expression during epithelial wound healing in corneas of vitamin A-deficient mice.

The purpose of this study is to investigate the expression of thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, during re-epithelialization in wounded corneas of vitamin A-deficient mice. Epithelial defects were created in the corneas of normal and Vitamin A-deficient mice with a microgrinder. Wounded corneas were stained with fluorescein and photographed for evaluation of re-epithelialization. Histological examination and immunohistochemical analysis of TSP-1 expression were also performed on the specimens from wounded corneas. In vitamin A-deficient mice, re-epithelialization of the wounded corneal epithelium was significantly delayed compared with that in normal mice. TSP-1 was detectable neither in the unwounded corneal epithelium of normal mice nor in that of vitamin A-deficient mice. In normal mice, linear staining of TSP-1 was observed on the wounded corneal surface and stroma at 30 min and 8 h to 16 h, respectively, after abrasion, and this TSP-1 expression disappeared at 36 to 48 h, when re-epithelialization was completed. In contrast, no TSP-1 staining was observed in the wounded corneas of vitamin A-deficient mice, except for the endothelial cells, throughout the wound healing process. Histological examination revealed a progressive increase in polymorphonuclear neutrophil infiltration in the stroma of the corneas of vitamin A-deficient mice during the healing process. These findings suggest that vitamin A may modulate the expression of TSP-1 in the corneas to accelerate the re-epithelialization of wounded corneas.

Expression of thrombospondin-1 is correlated with microvessel density in gastric carcinoma.

Thrombospondin-1 (TSP-1) has been shown to play a role in angiogenesis in a variety of cancers, but some studies indicated a difference in the mechanism of TSP-1 on neovascularization according to organ or histological type. Wild-type p53 protein has been shown to induce TSP-1 expression. We examined the expression of TSP-1 protein in 80 gastric carcinomas using immunohistochemistry and studied the relationship with microvessel counts, p53 expression and clinicopathological factors. We also performed reverse-transcriptase polymerase chain reaction analysis for the TSP-1 mRNA expression in gastric carcinoma cell lines and gastric cancer tissue after laser capture microdissection. Strong expression of TSP-1 protein was detected in 30 (38%) of the 80 cases. Positive staining for TSP-1 was seen in the cytoplasm of the cancer cells. TSP-1 mRNA expression was confirmed in a majority of gastric carcinoma cell lines and carcinoma tissues. Microvessel counts were significantly higher in tumors with strong TSP-1 protein expression than in those without expression or weak expression of TSP-1 ( P=0.011). No significant correlation was found between TSP-1 expression and p53 staining and clinicopathological factors. Our results support an idea that increased TSP-1 expression may be associated with an angiogenic phenotype in gastric carcinoma and suggest that TSP-1 may play diverse roles in each organ.

Thrombospondin 1 protein expression relates to good prognostic indices in ductal carcinoma in situ of the breast

Aim: Angiogenesis plays an important role in tumour growth and has been shown to occur around both in situ and invasive tumours. The degree of angiogenesis within tumours depends on...

Up-regulation of Matrix Metalloproteinase 9 by Thrombospondin 1 in Gastric Cancer

Background. Matrix metalloproteinase 9 (MMP-9) plays a key role in tumor cell invasion. It was recently reported that plasma levels of MMP-9 in patients with gastric cancer correlate with the tumors' metastatic potential. We previously demonstrated that thrombospondin 1 (TSP-1) up-regulates MMP-9 expression by endothelial cells and promotes tumor cell invasion. We hypothesized that TSP-1 plays a role in the up-regulation of MMP-9 in gastric cancer. Methods. MMP-9, TSP-1, and CSVTCG-specific TSP-1 receptor expression were measured by immunohistochemical staining in 31 consecutive gastric adenocarcinomas from patients who did not undergo neoadjuvant chemotherapy or radiation therapy. Additionally, we measured TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 expression by Western blotting, zymography, and immunohistochemical staining in AGS gastric adenocarcinoma cells. We also investigated the effect of TSP-1 on MMP-9 expression by AGS cells. Results. TSP-1 localized to the tumor-associated extracellular matrix. CSVTCG-specific TSP-1 receptor and MMP-9 colocalized to tumor cells, fibroblasts, and tumor-associated microvessels. Intense staining for TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 correlated with markers of aggressive tumor behavior. AGS gastric adenocarcinoma cells expressed high levels of CSVTCG-specific TSP-1 receptor but not TSP-1. TSP-1 up-regulated MMP-9 expression by AGS cells. Conclusion. We conclude that TSP-1 plays a role in the up-regulation of MMP-9 expression in gastric cancer. Our data also suggest a correlation between expression of TSP-1, CSVTCG-specific TSP-1 receptor, and MMP-9 and the acquisition of an aggressive tumor phenotype.

Reduced thrombospondin-1 at presentation predicts disease progression in superficial bladder cancer.

Superficial bladder cancer (SBC) presents a difficult clinical dilemma at diagnosis as only a small subgroup of patients will subsequently develop invasive disease. Study of cancer biology has found that angiogenesis is central to growth and spread. This study examines the relationship between the angiogenic inhibitory factor Thrombospondin-1 (TSP-1) at initial presentation and subsequent progression of SBC. Using immunohistochemistry, 220 cases of SBC were examined for pattern and extent of expression of TSP-1 at initial presentation. TSP-1 was detected in perivascular tissue, at the epithelial-stromal junction, in the stroma and in tumour cells and reduced perivascular TSP-1 staining at presentation was an independent predictive factor for the subsequent development of muscle invasive or metastatic disease. This adds further weight to the theory that TSP-1 plays a major part in the biology of bladder cancer possibly through the control of angiogenesis.

Expression of thrombospondin-1 in human pancreatic adenocarcinomas: role in matrix metalloproteinase-9 production.

Human pancreatic adenocarcinoma, an aggressive malignant disease, shows a strong desmoplastic reaction characterized by a remarkable proliferation of interstitial connective tissues. Thrombospondin-1 (TSP-1), a 450 kDa platelet and matrix glycoprotein, has been implicated in tumor invasion, angiogenesis and metastasis. TSP-1 and MMP-9 expression in pancreatic adenocarcinoma and control pancreas tissues was measured by immunohistochemistry. TSP-1 expression in pancreatic carcinoma cell lines, fibroblasts, and endothelial cells was measured by a competitive TSP-1 enzyme linked immunosorbent assay (ELISA). The effect of TSP-1 on MMP-9 production in pancreatic carcinoma cell lines was measured by zymography and Western blot analysis. Eighty five per cent (23/27) of cases of pancreatic adenocarcinoma showed increased TSP-1 staining in the desmoplastic stroma adjacent to tumor cells. No specific positive staining for TSP-1 was observed in the normal pancreatic tissues and the inflammatory areas. TSP-1 localized in tumor stroma surrounding the tumor cells expressing MMP-9. Using TSP-1 competitive ELISA, the secretion of TSP-1 by different pancreatic cancer cell lines into culture medium varied from 11.45 plus minus 14.08 to 275.82 plus minus 45.56 ng/10 6 cells/24 hours. The amounts of TSP-1 detected in both culture media and cell extracts from fibroblasts or endothelial cells were at least 2-3 fold higher than those from pancreatic cancer cells. TSP-1 augmented the production of matrix metalloproteinase-9, a matrix degrading enzyme, in pancreatic cancer cells in vitro. Stromally-derived TSP-1 up-regulates the production of MMP-9 by pancreatic adenocarcinoma. These data are consistent with the conclusion that TSP-1-rich stroma is involved in regulating matrix remodeling in tumor invasion.

Thrombospondin-1 Expression in Epithelial Ovarian Carcinoma: Association with p53 Status, Tumor Angiogenesis, and Survival in Platinum-Treated Patients

Objective. The regulation of the metastatic process in epithelial ovarian cancer has not been well defined. Similar to other tumor types, the angiogenic phenotype in ovarian cancer strongly influences clinical outcome, suggesting that the acquisition of a pro-angiogenic environment is essential to the process of ovarian cancer proliferation and metastasis. Thrombospondin-1 (TSP-1) is a potent peptide shown in other tumor systems to be associated with angiogenesis and possibly regulated by p53, a gene which is mutated in as high as 50% of advanced ovarian cancers. The purpose of this study was to investigate TSP-1 expression in invasive epithelial ovarian cancer and to examine the relationship between TSP-1 expression and the degree of angiogenesis. In addition, we examined whether TSP-1 expression was associated with overexpression of p53.Methods. Frozen sections obtained from 85 patients with invasive epithelial ovarian cancer were examined immunohistochemically for expression of TSP-1 and p53. The sections were examined microscopically by two investigators, who were blinded to the clinicopathologic variables. Outcome variables included the correlation among TSP-1, angiogenesis, and p53, as well as the association between TSP-1 expression and survival.Results. The majority (62%) of cases demonstrated high levels (3+) of TSP-1 expression; 7% demonstrated no TSP-1 expression. p53 was overexpressed in 55% of cases, and expression was inversely correlated with TSP-1 staining. Thirteen cancers had 0 or 1+ TSP-1 staining; 12 (92%) of these overexpressed the p53 protein. In contrast, only 49% of tumors with high expression of TSP-1 have overexpression of p53 (P = 0.02). TSP-1 was suggestive for improved survival in patients with advanced disease; high TSP-1 expression was associated with a median survival of 2.4 years compared to 1.5 years for patients with tumors having a lower degree of TSP-1 expression (P = 0.06).Conclusion. These data suggest that TSP-1 may possess a tumor inhibitory function in patients with advanced epithelial ovarian carcinoma. The reduction of TSP-1 expression associated with overexpression of p53 may be coupled with the development of a pro-angiogenic environment and malignant phenotype.

Impaired angiogenesis in the aging kidney: Vascular endothelial growth factor and thrombospondin-1 in renal disease

<h2>Abstract</h2> We investigated the relationship of changes in the microvasculature to age-related structural and functional changes in the kidney to determine whether there was evidence of impaired angiogenesis and whether the loss of microvasculature could be accounted for by changes in the local production of angiogenic or antiangiogenic factors. Glomerular and peritubular capillary number, density, and endothelial cell proliferation were determined in aging (24 months; n=9) and young (3 months; n=8) rat kidneys and correlated with renal functional and structural changes and alterations in renal expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aging rats showed a focal decrease in both peritubular capillary (peritubular capillary staining, 5.4% ± 1.8% versus 11.3% ± 2.0% per 100 tubules; rarefaction index, 10.6% ± 4.6% versus 0.6% ± 0.1%, aging versus young rats; <i>P</i> < 0.05 and <i>P</i> < 0.001, respectively) and glomerular capillary loops (27.3 ± 6.9 versus 50.7 ± 7.4/glomerulus, aging versus young rats; <i>P</i> < 0.001). The number of proliferating endothelial cells was decreased in aging rats compared with young rats (glomerular, 0.04 ± 0.01 versus 0.15 ± 0.03 positive cells/glomerular cross-section; peritubular, 0.7 ± 0.2 versus 4.3 ± 2.6 positive cells/mm²; <i>P</i> < 0.05). In the aging kidney, VEGF expression was focally increased in the cortex compared with young rats, whereas a profound decrease was observed in the outer and inner medulla (total area of VEGF expression, 19.2% ± 11.4% versus 39.3% ± 7.6%; <i>P</i> < 0.05). Tubular VEGF expression correlated with peritubular capillary density (<i>r²</i> = 0.57; <i>P</i> < 0.01) and inversely correlated with tubular osteopontin (<i>r²</i> = –0.55; <i>P</i> < 0.05) and macrophage infiltration (<i>r²</i> = –0.64; <i>P</i> < 0.01). TSP-1 staining was increased in the glomeruli and tubulointerstitium of the aging rats. Glomerular TSP-1 score correlated inversely with glomerular capillary number (<i>r²</i> = –0.89; <i>P</i> < 0.001). Tubulointerstitial TSP-1 also correlated with percentage of positive staining of peritubular capillary (<i>r²</i> = –0.59; <i>P</i> < 0.001). Glomerular capillary number showed significant correlation with glomerulosclerosis score, as well as with 24-hour urinary protein excretion. Peritubular capillary density also inversely correlated with interstitial fibrosis score and urinary protein excretion. In conclusion, glomerular and peritubular capillary loss in the aging kidney correlate with alterations in VEGF and TSP-1 expression and also with the development of glomerulosclerosis and tubulointerstitial fibrosis. These findings suggest that impaired angiogenesis associated with progressive loss in renal microvasculature may have a pivotal role in age-related nephropathy.

Impaired angiogenesis in the aging kidney: Vascular endothelial growth factor and Thrombospondin-1 in renal disease

We investigated the relationship of changes in the microvasculature to age-related structural and functional changes in the kidney to determine whether there was evidence of impaired angiogenesis and whether the loss of microvasculature could be accounted for by changes in the local production of angiogenic or antiangiogenic factors. Glomerular and peritubular capillary number, density, and endothelial cell proliferation were determined in aging (24 months; n = 9) and young (3 months; n = 8) rat kidneys and correlated with renal functional and structural changes and alterations in renal expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aging rats showed a focal decrease in both peritubular capillary (peritubular capillary staining, 5.4% ± 1.8% versus 11.3% ± 2.0% per 100 tubules; rarefaction index, 10.6% ± 4.6% versus 0.6% ± 0.1%, aging versus young rats; P < 0.05 and P < 0.001, respectively) and glomerular capillary loops (27.3 ± 6.9 versus 50.7 ± 7.4/glomerulus, aging versus young rats; P < 0.001). The number of proliferating endothelial cells was decreased in aging rats compared with young rats (glomerular, 0.04 ± 0.01 versus 0.15 ± 0.03 positive cells/glomerular cross-section; peritubular, 0.7 ± 0.2 versus 4.3 ± 2.6 positive cells/mm2; P < 0.05). In the aging kidney, VEGF expression was focally increased in the cortex compared with young rats, whereas a profound decrease was observed in the outer and inner medulla (total area of VEGF expression, 19.2% ± 11.4% versus 39.3% ± 7.6%; P < 0.05). Tubular VEGF expression correlated with peritubular capillary density (r2 = 0.57; P < 0.01) and inversely correlated with tubular osteopontin (r2 = –0.55; P < 0.05) and macrophage infiltration (r2 = –0.64; P < 0.01). TSP-1 staining was increased in the glomeruli and tubulointerstitium of the aging rats. Glomerular TSP-1 score correlated inversely with glomerular capillary number (r2 = –0.89; P < 0.001). Tubulointerstitial TSP-1 also correlated with percentage of positive staining of peritubular capillary (r2 = –0.59; P < 0.001). Glomerular capillary number showed significant correlation with glomerulosclerosis score, as well as with 24-hour urinary protein excretion. Peritubular capillary density also inversely correlated with interstitial fibrosis score and urinary protein excretion. In conclusion, glomerular and peritubular capillary loss in the aging kidney correlate with alterations in VEGF and TSP-1 expression and also with the development of glomerulosclerosis and tubulointerstitial fibrosis. These findings suggest that impaired angiogenesis associated with progressive loss in renal microvasculature may have a pivotal role in age-related nephropathy.

Significant correlation between thrombospondin 1 and serine proteinase expression in rheumatoid synovium.

Thrombospondin 1 (TSP1) is a potent active site inhibitor of leukocyte elastase and cathepsin G. This effect is markedly dependent on the disulfide-bond conformation of TSP1, with one isoform, TSP1(0.1), being the most potent. The aims of this study were to examine the expression of different disulfide-bonded isoforms of TSP1 in inflammatory environments in which elastase and cathepsin G are present in variable amounts, and to determine the relationship between these proteinases and their potential inhibitor. Immunohistochemical staining and histomorphometric analysis were used to examine adjacent sections of synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and meniscal trauma (MT), for expression of TSP1 and the TSP1(0.1) isoform, elastase, cathepsin G, and chymase. TSP1 localized to vessels and cells within the synovium. TSP1 expression was highly up-regulated in RA (mean density 98 cells and vessels/mm2, compared with 13/mm2 in OA and 17/mm2 in MT). The TSP1(0.1) isoform was found virtually exclusively in RA, with 44% of vascular TSP1 staining being due to the TSP1(0.1) isoform in RA, as compared with 7% in OA (P = 0.0047). Elastase- and cathepsin G-positive cells were abundant in RA, with mean densities of 106 cells/mm2 and 103 cells/mm2, respectively, compared with 2 cells/mm2 and 11 cells/mm2 in OA. There was a wide range of both TSP1 and proteinase expression within the RA group, but samples containing large numbers of elastase- and cathepsin G-positive cells also showed high expression of TSP1, especially TSP1(0.1). A strong correlation was found between elastase or cathepsin G densities and TSP1(0.1) expression in blood vessels (r = 0.86 and r = 0.76 respectively, P < 0.01). TSP1(0.1), with the most potent inhibitory activity in vitro, is specifically up-regulated in RA, and this up-regulation is in proportion to the numbers of surrounding leukocytes containing elastase and cathepsin G. One role of TSP1 may be to act as a matrix-based regulator of leukocyte-derived serine proteinases in vivo.